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Biomedical subjects

R Hampp

Publications and source records attributed to R Hampp.

At least 19 recordsLinked to original sources

Carbon dioxide concentration and nitrogen input affect the C and N storage pools in Amanita muscaria-Picea abies mycorrhizae.

We studied the influence of elevated atmospheric CO2 concentration ([CO2]) on the vacuolar storage pool of nitrogen-containing compounds and on the glycogen pool in the hyphal sheath of Amanita muscaria (L. ex Fr.) Hooker-Picea abies L. Karst. mycorrhizae grown with two concentrations of ammonium in the substrate. Mycorrhizal seedlings were grown in petri dishes on agar containing 5.3 or 53 mg N l(-1) and exposed to 350 or 700 microl CO2 l(-1) for 5 or 7 weeks, respectively. Numbers and area of nitrogen-containing bodies in the vacuoles of the mycorrhizal fungus were determined by light microscopy linked to an image analysis system. The relative concentration of nitrogen in the vacuolar bodies was measured by electron energy loss spectroscopy (EELS). Glycogen stored in the cytosol was determined at the ultrastructural level by image analysis after staining the sections (PATAg test). Shoot dry weight, net photosynthesis and relative amounts of N in vacuolar bodies were greater at the higher N and CO2 concentrations. The numbers and areas of vacuolar N-containing bodies were significantly greater at the higher N concentration only at ambient [CO2]. In the same treatment the percentage of hyphae containing glycogen declined to nearly zero. We conclude that, in the high N/low [CO2] treatment, the mycorrhizal fungus had an insufficient carbohydrate supply, partly because of increased amino acid synthesis by the non-mycorrhizal rootlets. When [CO2] was increased, the equilibrium between storage of glycogen and N-containing compounds was reestablished.

Amanita↗

Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems.

Ectomycorrhizas, the dominating mycorrhizal symbiosis in boreal, temperate and some tropical forests, are formed by 5000-6000 species of the asco- and basidiomycetes. This high diversity of fungal partners allows optimal foraging and mobilisation of various nitrogen and phosphorus forms from organic soil layers. In this review, two approaches to study the functioning of this multitude of symbiotic associations are presented. On selected culture models, physiological and molecular investigations have shown that the supply of hexoses has a key function in controlling the plant-fungus interaction via partner-specific regulation of gene expression. Environmental factors which affect fungal carbon supply, such as increased nitrogen availability, also affect mycorrhiza formation. Based on such laboratory results, the adaptative capability of ectomycorrhizas to changing field conditions is discussed. The second approach consists of analysing the distribution of mycorrhizas in ecosystem compartments and to relate distribution patterns to variations of ecological factors. Recent advances in identification of fungal partners in ectomycorrhizas by analysing the internal transcribed spacer of ribosomal DNA are presented, which can help to resolve sampling problems in field studies. The limits of the laboratory and the field approaches are discussed. Despite some problems, this combined approach is the most promising. Direct investigation of gene expression, which has been introduced for soil bacteria, will be difficult in the case of mycorrhizal fungi which constitute organisms with functionally varying structures.

Ascomycota↗

Carbon allocation in ectomycorrhizas: identification and expression analysis of an Amanita muscaria monosaccharide transporter.

Ectomycorrhizas are formed between certain soil fungi and fine roots of predominantly woody plants. An important feature of this symbiosis is the supply of plant-derived carbohydrates to the fungus. As a first step toward a better understanding of the molecular basis of this process, we cloned a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. Degenerate oligonucleotide primers were designed to match conserved regions from known fungal sugar transporters. A cDNA fragment of the transporter was obtained from mycorrhizal mRNA by reverse transcription-polymerase chain reaction. This fragment was used to identify a clone (AmMst1) encoding the entire monosaccharide transporter in a Picea abies/A. muscaria mycorrhizal cDNA library. The cDNA codes for an open reading frame of 520 amino acids, showing best homology to a Neurospora crassa monosaccharide transporter. The function of AmMST1 as monosaccharide transporter was confirmed by heterologous expression of the cDNA in a Schizosaccharomyces pombe mutant lacking a monosaccharide uptake system. AmMst1 was constitutively expressed in fungal hyphae under all growth conditions. Nevertheless, in mycorrhizas as well as in hyphae grown at monosaccharide concentrations above 5 mM, the amount of AmMst1 transcript increased fourfold. We therefore suggest that AmMst1 is upregulated in ectomycorrhizas by a monosaccharide-controlled mechanism.

Amanita↗

Fusion and metabolism of plant cells as affected by microgravity.

Plant cell protoplasts derived from leaf tissue of two different tobacco species (Nicotiana tabacum., N. rustica L.) were exposed to short-term (sounding rocket experiments) and long-term (spacelab) microgravity environments in order to study both (electro) cell fusion and cell metabolism during early and later stages of tissue regeneration. The period of exposure to microgravity varied from 10 min (sounding rocket) to 10 d (space shuttle). The process of electro fusion of protoplasts was improved under conditions of microgravity: the time needed to establish close membrane contact between protoplasts (alignment time) was reduced (5 as compared to 15 s under 1 g) and numbers of fusion products between protoplasts of different specific density were increased by a factor of about 10. In addition, viability of fusion products, as shown by the ability to form callus, increased from about 60% to more than 90%. Regenerated fusion products obtained from both sounding-rocket and spacelab experiments showed a wide range of intermediate properties between the two parental plants. This was verified by isozyme analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). In order to address potential metabolic responses, more general markers such as the overall energy state (ATP/ADP ratio), the redox charge of the diphosphopyridine nucleotide system (NADH/NAD ratio), and the pool size of fructose-2,6-bisphosphate (Fru 2,6 bisp), a regulator of the balance between glycolysis and gluconeogenesis, were determined. Responses of these parameters were different with regard to short-term and long-term exposure. Shortly after transition to reduced gravitation (sounding rocket) ratios of ATP/ADP exhibited strong fluctuation while the pool size of NAD decreased (indicating an increased NADH/NAD ratio) and that of Fru 2,6 bisp increased. As similar changes can be observed under stress conditions, this response is probably indicative of a metabolic stress compensation. Samples taken for up to 7 d of exposure to microgravity showed the opposite effect. Here, the ratios of ATP/ADP and of NADH/NAD, and the pool size of Fru 2,6 bisp were decreased. We take this an an indication of metabolic relaxation, i.e. decreased metabolic turnover. As rates of protoplast regeneration and cell division were obviously similar to 1-g controls, we conclude that under conditions of microgravity regenerating tobacco mesophyll protoplasts need less metabolic energy for the same effort.

Adenine Nucleotides↗

Regeneration of plant cell protoplasts under microgravity: investigation of protein patterns by SDS-PAGE and immunoblotting.

As part of the D-2 Spacelab mission, tobacco (Nicotiana tabacum L.) protoplasts were cultured for 10 days in microgravity and successfully regenerated into microcalli, which, after further cultivation on the ground, gave rise to intact plants. Protein analysis was performed on samples taken during the initial microgravity period and compared to ground controls. Total protein content and protein patterns were monitored, as well as the cytoskeletal proteins tubulin and actin, a key enzyme of secondary metabolism, phenylalanine ammonia lyase, and the pathogenesis-related protein osmotin. None of the investigated proteins showed a gravity-dependent effect. Since relative changes due to culture age were detectable in the immunoblots as well as in the total protein pattern, an adaptation of the cells to microgravity without major modifications of their protein complement may be assumed.

Actins↗

Electrofusion of plant cell protoplasts under microgravity--a D-2 spacelab experiment.

Plant cell protoplasts, derived from sexually incompatible plant species, have proved to be a good system for somatic hybridization by electrofusion. Under microgravity, an increase in fusion yield can be expected, especially if the parental cells differ markedly in size or specific density. On the D-2 spacelab mission flown in 1993, electrofusion experiments were performed with three different objects, i.e. tobacco as model system, Helianthus as an important crop, and Digitalis as a plant of pharmacological interest. The resulting fusion products were cultivated (along with parental cells) for 10 days under microgravity, and subsequently regenerated on ground for biochemical analysis. Results are presented on the observation of the fusion process during flight, heterofusion yields, ultrastructural investigation of fusion products immediately after fusion, and characterization of the resulting hybrids. The results are interpreted on the background of earlier microgravity-experiments on sounding rockets or parabolic flights.

Cardenolides↗

A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances.

A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.

Amanita↗

Suspensions of plant cells in microgravity.

Electrofusion of evacuolated with vacuolated mesophyll protoplasts of Nicotiana spec. was performed as part of the German Sounding Rocket Program (TEXUS). The results indicate a significant increase not only in the yield of 1:1 fusion products, but also in homo- and multifusion products. Heterokaryons obtained under microgravity have been shown to be viable to a higher degree with respect to their ability for light-dependent oxygen evolution (independent of other substrates than bicarbonate). Furthermore we were able to regenerate hybrid plants from suspensions of vacuolated and evacuolated protoplasts which were pulse-treated under microgravity. These expressed characteristics intermediate to those of the parental plants (Nicotiana tabacum (cv. Samsun), evacuolated; N. rustica, vacuolated).

Cell Fusion↗

Electric pulse induced membrane permeabilization. Spatial orientation and kinetics of solute efflux in freely suspended and dielectrophoretically aligned plant mesophyll protoplasts.

Asymmetric breakdown (occurring in only one hemisphere of the cell) was induced in freely suspended and dielectrophoretically aligned vacuole-containing or evacuolated plant protoplasts as well as in isolated vacuoles. In suspended cells breakdown was restricted to the hemisphere facing the anode and in isolated vacuoles to the opposite hemisphere. This difference in the orientation of the asymmetric breakdown can be explained by the opposite direction of the intrinsic membrane potentials of isolated vacuoles and of cells on which the generated potential difference is superimposed. The ensuing permeabilization of the membrane was microscopically monitored by dye uptake and by release of chloroplasts and of cytoplasmic and/or vacuolar solutes. The asymmetric release of intracellular substances (organic acids and/or amino acids) was detected by accumulation of chemotactic bacteria (Pseudomonas aeruginosa) close to the permeabilised membrane area of the cells or vacuoles. Maximum bacteria accumulation required about 5 min and subsequently disappeared after a further 20 min presumably because of the restoration of the original membrane impermeability. With vacuoles retention of the accumulated bacteria was shorter indicating that the resealing process of the tonoplast membrane was faster than that of the plasmalemma. From the kinetics of bacteria accumulation and retention it is therefore possible to deduce information about the life-span and the resealing properties of electropermeabilized membrane areas on the single-cell level. Symmetric breakdown in both hemispheres of the cells could be achieved by electric field-mediated cell rotation of about 180 degrees between two pulses of the same polarity or by application of two pulses of alternating polarity. In dielectrophoretically aligned protoplasts of comparable diameter, breakdown occurred in both hemispheres, even though the breakdown was still asymmetric. It could be demonstrated by the uptake of the vital dye neutral red that the size of the membrane area which was permeabilized was much larger in that hemisphere oriented to the anode than in the other one. The relevance of these observations for further improvement of electroinjection of macromolecules and of electrofusion is discussed. In particular, it is pointed out that positioning of differently sized cells in electric field-mediated hybridisation and the polarity of the breakdown pulse is of great importance with respect to hybrid yield.

Cell Membrane Permeability↗

Effects of microgravitation on electrofusion of plant cell protoplasts.

Electrofusion of evacuolated with vacuolated mesophyll protoplasts of Nicotiana tabacum was performed as part of the German Sounding Rocket Program (TEXUS 17, 1988). The results indicate a significant increase not only in the yield of 1:1 hybrids, but also in homo- and multifusion products. Hybrids obtained under microgravity have been shown to be viable to a higher degree with respect to their ability for light-dependent O2-evolution (independent of other substrates than bicarbonate). This finding is of interest for fusion experiments where only limited numbers of fusion partners are available (e.g. protoplasts from embryogenic tissues) or where fusion yields are extremely low under 1 x gravity (e.g. protoplasts of different specific density).

Cell Fusion↗

Appearance of photochemical function in prothylakoids during plastid development.

1. A method to separate the vesicles of prothylakoids from prolamellar body preparations obtained from etiolated and rapidly greening Avena laminae (0.25--4 h illumination ) is described. The prothylakoid preparations were found to be free from contaminating prolamellar bodies but enriched prolamellar body preparations (enriched prolamellar body preparations) still contained some adhering prothylakoid material. 2. Only existing beta-carotene appears to be transferred from the prolamellar bodies to the prothylakoids during early development and this ceases when freshly synthesized beta-carotene becomes available. 3. Prolamellar body structures proper show no positive association of existing or developing photochemical activities; these are only to be found in the developing prothylakoids. 4. Using methylviologen-linked electron transport-dependent oxygen consumption, Photosystem I activities may be detected with added diaminodurene within 15 min of illumination and within 30 min and 1 h with added tetramethylphenylenediamine and dichlorophenolindophenol, respectively. 5. During the 2nd, and 3rd. h of greening, proton-pumping capability and later ATP formation increased in prothylakoids in the presence of diaminodurene. 6. The first indications of Photosystem II activity using diphenylcarbazide as electron donor are shown at a similar time (2 h) with prothylakoids. The last photochemical activity to appear is the capacity to split water (3 h) and consequently the diphenylcarbazide activity diminished to zero before 8 h of illumination have passed. 7. The lack of effect of uncouplers such as NH4+ prior to 2 h suggests that in spite of some proton-pumping ability there is the possibility of proton-leaky areas existing within prothylakoids. This lack of a persistent proton gradient before 2 h of illumination may explain the different starting times of phenazine methosulfate- and diaminodurene-dependent photophosphorylation (0.25 and 2 h, respectively).

Carotenoids↗

Uptake of mevalonate and acetate during plastid development.

1. The envelopes of etioplasts and 1-2h etiochloroplasts are permeable to mevalonate, but plastids from etiolated tissue illuminated for longer than 4h show progressive impermeability towards mevalonate. 2. Acetate permeates the envelopes of 1-4h etiochloroplasts but does not significantly cross the envelopes of etioplasts or 8-24h etiochloroplasts. 3. A translocator system exists within the plastid envelopes for mevalonate which relies on malate as a counter-exchange anion.

Acetates↗

Lead and plants.

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Absorption↗